1. Field of the Invention
The present invention relates to a recombinant canine distemper virus which is useful as a vaccine against canine distemper and leishmaniasis, a vaccine comprising the recombinant virus, and an antiserum obtained from an animal inoculated with the vaccine.
2. Description of the Prior Art
Canine distemper occurs naturally in animals such as the families Canidae and Mustelidae. It is characterized by symptoms such as fever, respiratory disorders such as pneumonia and bronchitis, diarrhea and nervous disorders. This disease is one of the representative infectious diseases in canines. It has been controlled for about thirty years by inoculation with live vaccines, but recently it has been reported that the occurrence of this disease is increasing worldwide, including in Japan. Even vaccinated dogs sometimes develop the disease, indicating that there is a possibility that the prior art vaccination cannot completely control the currently widespread field isolates. It has been reported that when examining the cross neutralizing reaction of the serum obtained from infected dogs against the vaccine strain, the reactivity against the vaccine strain was low. Recently isolated field isolates have mutations in the H gene encoding membrane protein, which is important for infection. Dogs are used as an experimental animal for physiological or pharmaceutical studies, as a disease model for humans, and for studies of infectious diseases, and they are recognized to be useful to industry. Also, dogs have played an important role in the social life of humans as domestic animals for thousands of years. Therefore, the protection of dogs from canine distemper is desired eagerly in many fields.
Canine Distemper Virus (CDV) that causes canine distemper belongs to the genus Morbillivirus of the family Paramyxoviridae. The canine distemper virus particle is pleomorphic, and has the genomic RNA which does not serve as a template for translation, namely a negative single strand RNA, surrounded by an envelope of 150-200 nm in diameter.
The present inventors established a system for reconstituting the canine distemper virus, which system is necessary for gene manipulation of the virus (see Japanese Patent Application Kokai (Laid Open) No. 2001-275684). However, there remains a need for a multivalent vaccine against canine distemper virus and other pathogens to be actually prepared and for the efficacy thereof to be confirmed.
On the other hand, leishmaniasis is a zoonosis in humans and animals caused by Leishmania protozoa belonging to hemoflagellates, and it is designated as one of the infectious diseases which the World Health Organization intends to eradicate. The number of persons infected with this disease is estimated at about 12 million, and every year two million persons will be infected with this disease in 88 countries. Leishmaniasis is generally classified into three types of diseases, 1) visceral leishmaniasis, 2) cutaneous leishmaniasis, and 3) mucocutaneous leishmaniasis, and each is characterized by 1) abdominal symptoms such as splenohepatomegaly and diarrhea, anemia and leukopenia, 2) induration or tuberculation and ulceration of the skin, and 3) tissue destruction of the nasal septum, labia oris, palate, pharynx etc. Infection of humans occurs by entry of humans into the life cycle of the vector insect, sand fly, and dogs and wild canines or rodents acting as a host. Effective prophylaxis or therapy has not been developed. It has been reported that the elimination of the infection in dogs will allow the control of the infection of humans epidemiologically. Therefore, the development of vaccines or a method for prevention of the protozoa is expected to lead to the eradication of this infectious disease not only in the veterinary field but also in humans.
As mentioned above, there is currently no effective vaccine against leishimaniasis. Leishmania antigens such as LACK (Leishmania homologues receptors for activated C kinase), A2 and Promastigote Surface Protease (gp63) expressed in Leishmania are known as a target antigen for suppressing the propagation of Leishmania protozoa, and thereby promoting the cure of leishmaniasis. It is effective for protection against leishmaniasis to induce protective Th1 immune responses after the infection. A DNA vaccine which is characterized in that the LACK gene linked to a suitable transcriptional control sequence is inoculated directly into the body, and a recombinant vaccinia virus vaccine containing such a LACK gene have been reported to be effective in a vaccination experiment in mice and to be particularly more effective when used along with IL-12. A2 is one of the major antigen proteins expressed specifically to amastigotes of Leishmania protozoa (L. donovani) causing visceral leishmaniasis and is associated with the pathogenicity of leishmaniasis. In the vaccination experiment in mice, it has been reported that the inoculation of A2 leads to induction of protective Th1 immune responses. However, such vaccines are in the development stage and at present cannot be used practically.
The object of the present invention is to provide a recombinant bivalent virus vaccine which is effective to protect against canine distemper and leishmaniasis and allows a simple procedure in vaccination.
The present inventors noticed that since canine distemper virus grows essentially in a dog as a host and the vaccine comprising the virus induces an effective immune response and gives permanent immunity, the virus can be used as a virus vaccine vector, and they tried to construct recombinant canine distemper virus having a foreign gene involved in the prevention of the infection of Leishmania protozoa inserted therein. They found that the resulting recombinant canine distemper virus is effective as a vaccine against both canine distemper and leishmaniasis.
Thus, the present invention provides recombinant canine distemper virus containing a gene encoding a protein involved in protection against leishmaniasis inserted into the canine distemper virus genome.
The present invention also provides recombinant canine distemper virus which has the infectivity, and is capable of expressing a protein which leads to protection against leishmaniasis after vaccination with the recombinant virus within infected cells.
Furthermore, the present invention relates to the above-mentioned recombinant canine distemper viruses wherein at least one gene of the canine distemper virus genome, particulary a gene encoding a functional protein of canine distemper virus, is modified.
The above-mentioned recombinant canine distemper virus may contain at least one foreign gene in addition to a gene encoding a protein concerned with protection against leishmaniasis.
The present invention also relates to a bivalent vaccine against canine distemper virus and leishmaniasis, comprising the above-mentioned recombinant canine distemper virus. Furthermore, the present invention relates to an antiserum obtainable from bodily fluid which is recovered from the animal infected with the above-mentioned recombinant canine distemper virus.